The bg1G gene product is an RNA binding protein which functions as a transcriptional antiterminator in E. coli. We have identified the RNA target for this protein and now wish to study precisely how the protein interacts with the RNA. Preliminary indications are that the protein recognizes a region of RNA secondary structure with a bulged are being important. Other important regulators such as HIV Tat appear to also recognize such structures, however Bg1G protein does not seem to have a Tat-like RNA binding motif thus it may represent a novel family of RNA binding proteins. To study how Bg1G interacts with RNA we will purify the protein using a gene fusion approach since it will allow affinity purification. The purified protein will be used to study interaction with RNA by gel retardation and chemical footprinting. In parallel we will also purify a B.subtilis protein, SacY, which has properties similar to Bg1G and which shares significant homology with it. We will carry out a comparative series of binding studies to try to determine the elements within the proteins that are important for binding specificity. The SacY RNA target will be used in these studies as well since it differs in several respects from the Bg1G target. We have found that a specific mutation (C to A change) at position 50 in the Bg1G target RNA eliminates Bg1G recognition without altering the overall RNA structure. We intend to look for mutations in Bg1G that suppress the RNA alteration and thus hope to identify regions of contact. We will also search for mutants of Bg1G that have a dominant negative phenotype. These would provide additional support for the observation that Bg1G binds to RNA as a dimer. Finally we will look for mutations that alleviate any dominant negative effect with the aim of identifying sites important for dimerization or that interact with one another in the dimer.